Bifunctional Reagents for Formylglycine Conjugation: Pitfalls and Breakthroughs

Abstract

Dedicated to the memory of Thomas Dierks Formylglycine-generating enzymes specifically oxidize cysteine within the consensus sequence CxPxR to C α-formylglycine (FGly). This noncanonical electrophilic amino acid can subsequently be addressed selectively by bioorthogonal hydrazinoiso-Pictet-Spengler (HIPS) or Knoevenagel ligation to attach payloads like fluorophores or drugs to proteins to obtain a defined payload-to-protein ratio. However, the disadvantages of these conjugation techniques include the need for a large excess of conjugation building block, comparably low reaction rates and limited stability of FGly-containing proteins. Therefore, functionalized clickable HIPS and tandem Knoevenagel building blocks were synthesized, conjugated to small proteins (DARPins) and subsequently linked to strained alkyne-containing payloads for protein labeling. This procedure allowed the selective bioconjugation of one or two DBCO-carrying payloads with nearly stoichiometric amounts at low concentrations. Furthermore, an azide-modified tandem Knoevenagel building block enabled the synthesis of branched PEG linkers and the conjugation of two fluorophores, resulting in an improved signal-to-noise ratio in live-cell fluorescence-imaging experiments targeting the EGF receptor.

Bivalent EGFR-Targeting DARPin-MMAE Conjugates

“…Existing HIPS and Knoevenagel ligations have been further optimized by utilizing bifunctional HIPS and tandem Knoevenagel reagents in combination with highly efficient strain-promoted azide-alkyne cycloadditions (SPAAC) [ 39 , 40 ]. The new coupling strategy was tested with mono- and bivalent DARPins and a scFv construct targeting the EGFR.…”

“…Second, improved coupling efficiency was achieved by converting the genetically encoded FGly-tag CxPxR with formylglycine-generating enzyme (FGE) in combination with a novel coupling strategy. The relatively slow reaction of the formylglycine residue in a Knoevenagel reaction generating an azide-modified protein was combined with the fast strain-promoted azide-alkyne cycloaddition (SPAAC) [ 40 ] of the DBCO-modified drug monomethyl auristatin E (MMAE). Third, uptake and anti-tumor activity was analyzed in cellular assays and in a mouse xenograft model.…”

“…FGE oxidizes cysteine within the consensus sequence CxPxR to the non-canonical, electrophilic amino acid C α -formylglycine (FGly) [ 28 , 29 , 30 , 31 , 32 , 33 , 34 ], which can be addressed selectively by nucleophiles. In particular, hydrazine-functionalized indoles (Hydrazino- iso -Pictet-Spengler ligation) [ 35 , 36 , 37 , 38 , 39 ] or pyrazolones ( trapped and tandem Knoevenagel ligation) [ 40 , 41 , 42 , 43 ] are used in combination with FGE to label proteins or to produce antibody-drug conjugates.…”

Efficient Site‐Selective Immobilization of Aldehyde‐Tagged Peptides and Proteins by Knoevenagel Ligation

“…Therefore, the conversion was performed after expression and purification with three different FGE variants: soluble human FGE, soluble MtFGE and immobilized MtFGE (Scheme 2B). All resulted in quantitative FGly content, as shown by subsequent labeling with the aldehyde-reactive fluorophor 16 [38] (Figure 2B-2D). In this respect it is worth mentioning that the use of the immobilized FGE did not lead to contamination of the transaminase (Scheme 2D).…”

“…The Knoevenagel core segments 3 and 6, which are also commercially available, were synthesized as described in the literature. [36,38] Problems could be observed especially in the synthesis of 3 after deprotection by LiOH and acidic work-up. Due to the high polarity, the final separation of LiCl was only possible by multiple desalting steps with C18 silica.…”